journal

Preservation of boar semen at 18 ◦C induces lipid
peroxidation and apoptosis like
changes in spermatozoa

A. Kumaresan ∗, G. Kadirvel, K.M. Bujarbaruah 1, R.K. Bardoloi, Anubrata Das2, Satish Kumar, S. Naskar Artificial Insemination Laboratory, Division of Animal Production, ICAR Research Complex for NEH Region, Barapani, Umiam, Meghalaya 793103, India

Received 13 August 2007; received in revised form 10 January 2008; accepted 13 January 2008
Available online 18 January 2008

Abstract

Boar sperm functions, lipid peroxidation status, mitochondrial membrane potential (Ψm) and membrane permeability (apoptosis like features) were assessed during liquid preservation. Four ejaculates each from four Hampshire boars were extended with Beltsville Thawing Solution and preserved at 18 ◦C. At 0, 24, 48, 72 and 96 h of storage, each ejaculate was examined for sperm functions, lipid peroxidation, Ψm, and membrane permeability. The lipid peroxidation status of the spermwas assessed based on the malonaldehyde (MDA) levels. Detection ofΨm was done using 3,3-dihexyloxacarbocyanine iodide [DiOC6(3)]/propidium iodide (PI) assay and Yo-pro-1/PI assay was used to detect change in plasma membrane permeability. The sperm motility, viability and acrosomal integrity declined significantly (p < 0.05) from 0 to 96 h of preservation. At the start of the preservation, the MDA levels (nM/109 sperm) were low in sperm (99.83±2.69) and seminal plasma (191.98±11.58), which gradually increased up to the 96 h of storage. Highest negative correlation (r value) was observed between MDA levels and sperm motility (−0.97), live percent (−0.97), acrosomal integrity (−0.97) and hypo-osmotic sperm swelling test (HOSST) positive sperm percentage (−0.98). Strong positive correlation was observed between HOSST positive sperm percentage and intact acrosome percentage (r = 0.98). There was a significant (p < 0.05) increase in the sperm cells with low Ψm from 0 to 96 h of preservation. Before preservation, 14.85±4.66% of sperm cells of the ejaculate showed low mitochondrial membrane potential, whereas after 96 h of preservation, this proposition of cells increased up to 32.00±6.25%. The apoptotic sperm population was 8.33±2.31% in fresh semen, while this population was 25.19±4.25% at 96 h of preservation and the difference was significant (p < 0.05). The findings of the

∗ Corresponding author. Tel.: +91 364 2570362.
E-mail address: ogkumaresan@rediffmail.com (A. Kumaresan).
1 Animal Science Division, Krishi Bhavan, New Delhi, India.
2 National Research Centre on Pig, Guwahati, Assam, India.
0378-4320/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2008.01.006

journal

In vitro sperm penetration through the zona
pellucida of immature and in vitro matured oocytes
using fresh, chilled and frozen canine semen

Monica De los Reyes a,∗, Jaime Palomino a, Johanna de Lange a, Carla Anguita a, Claudio Barros ba Laboratory of Animal Reproduction, Faculty of Veterinary Sciences, University of Chile, Casilla 2 Correo 15, Santiago, Chile b Laboratory of Embriology, Faculty of Biological Sciences, P. Catholic University of Chile, Santiago, Chile Received 28 June 2007; received in revised form 5 December 2007; accepted 12 December 2007
Available online 28 December 2007

Abstract

The aim of this study was to evaluate the effect of sperm cryopreservation and the maturation state of the oocyte on the time course of canine gamete interaction during co-culture for periods of 1–10 h. Semen samples were obtained by digital stimulation and ejaculates processed as fresh, chilled and frozen samples. Sperm were co-cultured with immature or in vitro mature bitch oocytes for up to 10 h. At hourly intervals, oocytes were evaluated for sperm penetration with epifluorescence microscopy. The results were analyzed statistically using generalized linear models. Spermatozoa treatments had a significant effect on the total percentage of oocyte penetration for both types of oocytes; fresh spermatozoa showed the highest average penetration rate, while frozen sperm showed the lowest value (p < 0.05). At the 1st hour of co-culture, chilled and frozen dog sperm had a higher penetration percentage (p < 0.05) of in vitro matured canine oocytes (43.6% and 45.7%, respectively) than the fresh sperm had (33.8%). Sperm penetration was directly proportional to the time of incubation, when fresh or chilled sperm were used (P < 0.05); in contrast, frozen dog sperm did not change penetration rates with either immature or in vitro matured oocytes over time. Therewas a significant difference in the average of penetration rate between immature (47.3%) and in vitro matured oocytes (56.6%) throughout the 10 h of culturing; irrespective of sperm treatment. The optimal incubation time in terms of maximizing penetration rates probably are dependent on how spermatozoa were processed prior to fertilization.

© 2007 Elsevier B.V. All rights reserved.
Keywords: Capacitation; Cryopreservation; Dog sperm; Gamete interaction

∗ Corresponding author. Tel.: +56 2 9785534; fax: +56 2 9785611.
E-mail address: mdlreyes@uchile.cl (M. De los Reyes).
0378-4320/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.anireprosci.2007.12.010

journal

The 'beneficial' adipokines in reproduction and fertility

D B Campos^1,2, M-F Palin³, V Bordignon⁴ and B D Murphy¹

1. ¹Faculté de médecine vétérinaire, Centre de recherche en reproduction animale, Université de Montréal, Québec, Canada
2. ²Faculdade de Medicina Veterinária e Zootecnia, Departamento de Cirurgia, Universidade de São Paulo, Sao Paolo, Brasil
3. ³Dairy and Swine Research and Development Center, Agriculture and Agri-Food Canada, Lennoxville, Québec, Canada
4. ⁴Department of Animal Science, McGill University, Québec, Canada

Correspondence: Dr BD Murphy, Faculté de médecine vétérinaire, Centre de recherche en reproduction animale, Université de Montréal, CP 5000, 3200 rue Sicotte, St-Hyacinthe, Québec, Canada J2S7C6. E-mail: bruce.d.murphy@umontreal.ca

Received 8 April 2007; Revised 29 May 2007; Accepted 16 July 2007; Published online 9 October 2007.

Abstract

The objective of this study was to review the available information on the signaling proteins produced by adipose tissue in the context of their role in regulating reproductive processes, including ovarian and uterine function. It is well known that both obesity and excessive leanness are associated with reproductive dysfunction. Adipokines are cytokines predominately or exclusively expressed by adipose tissue that circulate and affect target tissues. Four known adipokines, adiponectin, visfatin/PBEF, omentin and vaspin, all increase tissue sensitivity to insulin, and are thus described as 'beneficial'. There is strong support for a role for adiponectin in the function of the ovary and placenta. There is evidence for direct effects of this adipokine on the late stages of folliculogenesis, and additive interactions of adiponectin with insulin and gonadotropins in inducing periovulatory changes in ovarian follicles. In addition, clinical and genomic studies associate hypoadiponectinemia with obesity-related reproductive disorders, including the polycystic ovarian syndrome. The roles for visfatin/PBEF, omentin and vaspin in reproduction remain to be established. The conclusion thus drawn is that the expression of insulin-sensitizing adipokines varies with adipose abundance. These adipokines have demonstrated both the potential effects on ovarian function and the possible effects on the formation of the placenta, acting through multiple mechanisms.

Keywords:

adipokines, adiponectin, insulin sensitivity, ovary, uterus, placenta